PAPER [Link].

org/analyst | The Analyst

An impedance array biosensor for detection of multiple antibody–antigen

interactions
Xiaobo Yu,a Renji Lv,b Zhenqiu Ma,b Zhihong Liu,b Yanhong Hao,a Qingzhang Lia and Danke Xu*b
Received 5th December 2005, Accepted 30th March 2006
First published as an Advance Article on the web 13th April 2006
DOI: 10.1039/b517148b

Electrochemical impedance spectroscopy (EIS) combined with a gold electrode array was
developed to detect multiple antibody–antigen interactions. Hepatitis B surface antigen (HBsAg),
as a model sample, was employed to evaluate the characteristics of the biosensor. The array
was fabricated by immobilizing antibodies on the self-assembled molecules surface of the
electrodes. The surface characteristics of the array during the binding process including the
antibody–antigen conjugation and the sandwich complex with HRP-labeled antibody,
as well as the precipitation layer, were characterized by atomic force microscopy (AFM) and
electrochemical impedance spectroscopy, respectively. A linear relationship between electron-
transfer resistance and the concentrations of HBsAg ranged from 10 pg ml21 to 1 ng ml21
and the detection limit of 10 pg ml21 was obtained. 100 pg ml21 antigen samples, such as rat
IgG, HBsAg and HBeAg, as well as the antigen mixture, were incubated with the relative
antibody-modified electrodes on the array. No obvious cross-talk reaction was observed. All
these results confirm the feasibility of applying electrochemical impedance spectroscopy to the
electrode array.

1. Introduction develop an electrochemical array biosensor that could be

employed to detect the multiple antibody–antigen interactions
With the development of research in post-genomics and while keeping its high sensitivity is still a challenge at present.
proteomics, more and more biomarkers will be discovered As an alternative, a sensitive electrochemical technique,
and applied to molecular diagnosis and drug development as electrochemical impedance spectroscopy, can monitor the
well as the practice of medicine. Protein detection techniques interface response of the electrode by applying a periodic
such as immunological sensors and protein microarrays play a small amplitude ac signal on the electrode. In this case, the
significant role because of their high sensitivity, high through- adsorption or desorption of insulating materials on conductive
put and automation.1–3 In general, electrochemical and optical supports can be assayed due to the change of the interfacial
techniques are two main detection approaches to immuno- electron transfer features at the electrode surface.5 Compared
sensors. Advantages of electrochemical methods are brief with above electrochemical methods, EIS can provide infor-
design, small dimensions, low power requirement and low cost. mation on the surface difference on the transducer before
Among the various electrochemical techniques, amperome- and after biological molecular interactions such as antigen–
try and potentiometry are two main methods. In these cases, antibody and protein–protein.6 In these years, EIS immuno-
the electrode is used both as an integrated recognition and a sensors have been utilized for determination of DNA,7
transduction element, in which capture molecules are immo- protein,8 microorganism,9 drug small molecules10 and cell
bilized on the electrode surface through a conjugation reaction apoptosis,11 etc. Furthermore, they have been extended to
and the recognition signal is monitored by the transducer. The array format for label-free detection of DNA aptamer–protein
enzyme label method is most often used for the detection interactions and protein–protein interactions in our labora-
system, in which the small redox molecules produced by tory, which holds their promising applications in high-
conjugated enzyme might diffuse onto the electrode surface throughput screening of protein–protein or DNA–protein
and then cause detectable signals. However, few electrochemi- interactions.12,13 On the other hand, how to amplify the signal
cal methods have been further developed to assay protein of the antigen–antibody binding event is always a relevant
array because the diffusion of these products would lead to question in the development of immunosensors. In previous
cross-talk interference.4 In addition, the modified layer, such reports, alkaline phosphatase (AP) labelled secondary anti-
as protein or antibody, would hinder a redox probe from body9 and horseradish peroxidase (HRP) GM1-functionalized
transferring into the electrode surface. As a result, how to liposomes14 had been developed to detect Escherichia coli
O157:H7 and the cholera toxin through catalyzing their
a
College of Veterinary Medicine, Northeast Agricultural University, substrates of 5-bromo-4-chloro-3-indolyl phosphate and
Harbin, 150030, China 4-chloro-1-naphthol, respectively. Enzyme-labelled secondary
b
Department of Biochemistry, Beijing Institute of Radiation Medicine, antibodies convert their substrates into enzymatic reaction
Beijing Proteomics Research Center, Beijing, 100850, People’s Republic
of China. E-mail: xudk@[Link]; Fax: +86-10-66932225; products that are insoluble precipitation deposited on the
Tel: +86-10-66932225 binding site of the antibody–antigen, which can amplify the

This journal is ß The Royal Society of Chemistry 2006 Analyst, 2006, 131, 745–750 | 745
binding signal of antibody–antigen and is readily detected with 2.2. Apparatus
EIS and a quartz crystal microbalance, respectively. Moreover,
Electrochemical impedance spectroscopy was all performed
enzyme labels could not only amplify signal, but also increase
on a CHI instruments Model 660 electrochemical analyzer
the difference between the sample and the control. To our
(CHI instruments Inc, USA). A two-electrode system consist-
knowledge, such a detection approach has not been developed
ing of a gold working electrodes array and a standard Ag/AgCl
for antibody arrays or array biosensors yet.
electrode served as both the counter and reference electrode.
In this experiment, a gold electrode array was fabricated and All atomic force microscopy (AFM) experiments were
detected by electrochemical impedance spectroscopy. Hepatitis performed using an SFM9500J3 (Shimadzu, JAPAN) and all
B surface antigen was first served as a model to evaluate AFM images were further analysed with Particle Analysis
the characteristics of the electrochemical array biosensor. Software (Shimadzu, Japan).
The electrode assembly and reaction process, including the
immobilization of the antibodies, the conjugation with the 2.3. Procedures
antigens as well as enzyme-labelled secondary antibodies,
and biocatalyzed precipitation on the electrode surface, was 2.3.1. Fabrication of gold electrodes array and immobilization
characterized by AFM and EIS, respectively. In addition, of the antibodies. One gold array consisting of four gold
goat-anti-rat IgG, anti-HBsAg antibody and anti-HBeAg working electrodes was fabricated by using vapour deposition
antibody were immobilized on the array electrodes and the of thin gold films (200 nm) with an underlayer of chromium
resulting antibody array was used to assay rat IgG, HBsAg, (50 Å) on the quartz substrate. The dimensions of each
HBeAg, and the mixture, respectively. single gold working electrode were 2 mm 6 2 mm. Prior to
the modification, the gold array was immersed in a piranha
solution (H2SO4–H2O2, 3:1) for 30 min and then cleaned
2. Experimental thoroughly using water and ethanol.
2.1. Reagents The whole immobilization and analytical protocol is
illustrated as Fig. 1. A home-constructed immobilization
2-Mercaptoethylamine and bovine serum albumin (BSA) were frame made from PDMS containing four wells was then
purchased from Sigma (St. Louis, MO). Glutaraldehyde aligned onto the array according to the pattern of the array
was purchased from Beijing Chemical Reagents Co. (Beijing, electrodes. After the frame had been attached to the array,
China). Poly(dimethylsiloxane) (PDMS) was purchased from 2-mercaptoethylamine (10 mg ml21) was injected to each well
Dow Corning Co. (Sylgard 184, Midland, MI). Hepatitis B and then kept for 4 h for the formation of self-assembled
virus surface antigen (HBsAg), anti-HBsAg antibody, HRP- monolayer (SAM), followed by the addition of glutaraldehyde
conjugated anti-HBsAg antibody, Hepatitis B e antigen (5%) for 1 h incubation. As a result, the resulting electrode
(HBeAg), anti-HBeAg antibody and HRP-conjugated anti- surface with aldehyde groups would readily couple with amino
HBeAg antibody were purchased from Kewei Diagnostic groups of the antibodies. Then, 10 mL of antibody solution
Reagents Co. (Beijing, China). Rat IgG (rIgG), Goat-anti- (1 mg ml21) was introduced into each well and allowed to
rIgG, HRP-labelled goat anti-rIgG and 3-amino-9-ethyl incubate overnight. After the antibody-modified array had
carbazole (AEC) were purchased from Sino-Am Biotech Co. been rinsed three times using 10 mmol L21 PBS containing
(Beijing, China). 0.02%Tween 20 (pH 7.4), the BSA (10 mg ml21) was used to
All of the other chemicals were of reagent grade and used as deactivate the excess aldehyde groups and block the electro-
received. Phosphate buffered saline (PBS) was used for the statically bound proteins. Finally, the frame was removed and
preparation of buffers. The dilution of HRP-conjugated anti- the resulting array was stored at 4 uC.
HBsAg antibody (l:5000) and HRP-conjugated anti-HBeAg
antibody (1:2500) was prepared with PBS before use. Aqueous 2.3.2. Analytical procedure. Prior to analysis, the reaction
solutions were prepared with deionized water. frame was mounted on the array to form an analytical pool.

Fig. 1 Schematic illustration of the immobilization and analytical protocol.

746 | Analyst, 2006, 131, 745–750 This journal is ß The Royal Society of Chemistry 2006
Then, 60 mL of sample solution was injected into the pool and antibody, (b) after incubation with 100 pg ml21 HBsAg,
kept for incubation for 1 hour, followed by a rinse with 10 mM (c) coated with sandwich complexes formed by addition of
PBS containing 0.02% Tween 20 (pH 7.4) three times. The HRP-labelled antibody, and (d) coated with the precipitation
resulting array was further reacted with HRP-labelled anti- produced by the enzymatic catalysis. Generally, the semi-
body for 1 h. After rinsing with 10 mM PBS three times, AEC circular portion observed at higher frequencies corresponds to
solution was added for development for 30 min. Finally, the electron-transfer limited process, whereas the linear part is
the AEC solution was removed and the array was rinsed characteristic of the lower frequency range and represents the
thoroughly with water. diffusion-limited electron-transfer process. As shown in Fig. 2,
When carrying out EIS measurements, 60 mL of 5 mmol L21 the semicircular diameter corresponding to the electron
[Fe(CN)6]42/32 in 10 mmol L21 PBS was pipetted into the transfer resistance at the electrode surface gradually increased
analytical pool, and an Ag/AgCl electrode was positioned into when the antibody-modified electrodes were incubated with
the pool to form a circuit. The Nyquist plot is expressed in the antigen as well as the HRP-labelled antibody. The results
term of a real (Zre) and an imaginary (Zim) component and the suggested that the thickness of the modified layer of the
frequency ranges from 0.1 Hz to 10 kHz at the formal potential electrode increased with the conjugation of the antigen and the
of 250 mV, using alternating voltage of 5 mV. All experiments formation of the sandwich complexes. Such a phenomenon
were performed at 25 uC. becomes more significant with the production of the AEC
precipitation under catalysis by the addition of labelled HRP.
2.3.3. AFM imaging. The assembly procedures of the This provides an approach to development of higher sensitivity
antibodies on the gold surface and the reaction with the detection for antibody–antigen interactions.
samples were the same as above. Before testing, the modified The assembly process of these molecules at the gold
electrode was rinsed with PBS containing 0.02%Tween 20 electrode surface was also proved by obtaining topological
(pH 7.4) and water to remove nonspecific absorbed molecules, images using AFM, shown as Fig. 3. Moreover, in order to
and dried in the clean air. All AFM experiments were obtain the average height of particles on the electrode surface,
performed using the tapping mode in open air. all AFM images were analysed with particle analysis software.
As shown in the histogram of Fig. 3, the x-axis and y-axis
3. Results and discussion represent the height and the number of particles, respectively.
It shows that the electrode surface was covered with a homo-
3.1. Characterization of the antibody-modified electrode geneous anti-HBsAg antibodies layer besides a few aggregates
In this study impedance spectroscopy was presented as of antibodies (Fig. 3(a)). Compared with antibody-modified
Nyquist plots, in which Zim and Zre represent the imaginary electrode, the average height of antibody–antigen complexes in
and real part of the impedance, respectively. Fig. 2 illustrates Fig. 3(b) does not show an obvious difference due to low
the results of impedance spectroscopy in the presence of concentration of HBsAg sample (100 pg ml21) as well as the
5 mmol L21 [Fe (CN)6]42/32 as redox probe in PBS obtained random orientation of antibodies. When HRP-labelled anti-
by the gold electrode (a) immobilized with anti-HBsAg HBsAg antibody was then introduced, some column-like
protein complex could be seen clearly from the electrode
surface and the increased height was about 2.6 nm, which is in
accordance with the previous report.15 It implies that the
sandwich complex was formed on the electrode, as shown in
Fig. 3(c). Fig. 3(d) shows the topological image of the
precipitation formed by catalysis of HRP-labelled anti-
HBsAg antibodies at the gold electrode surface. Owing to
precipitation, catalysed by HRP, labelled secondary antibody
could deposit on the binding site of antibody and antigen in
3 dimensions: the diameter and height of particles on the
electrode are increased significantly. As is shown in histogram
of Fig. 3(d), the average height of the particles at the electrode
surface increased to about 26 nm, which is almost four times
than that of sandwich complexes layer at the electrode surface.
The result implied that the precipitation could bring a large
increase of the thickness compared with that of merely
Fig. 2 Nyquist diagram (Zim versus Zre) for the faradaic impedance antibody–antigen complex at the electrode surface, which
measurement of the gold electrode in 10 mmol L21 PBS (pH 7.4) accordingly leads to rather large resistance for electronic
containing 5 mmol L21 [Fe (CN)6]42/32. (a) Immobilized with anti-
transfer of the redox probe. In fact, these results were in
HBsAg antibodies; (b) modified by antibody–antigen complexes after
accordance with those obtained by impedance measurement.
incubation with 100 pg ml21 HBsAg; (c) modified with sandwich
complexes by addition of HRP-labelled anti-HBsAg antibodies; (d)
While a small increase in Ret occurred after binding with the
coated with the enzymatic precipitation by introduction of AEC antigens (DRet = 9 6 102) and HRP-labelled antibody (DRet =
substrate. The impedance spectra were recorded within a frequency 6 6 102), the formation of the precipitation layer led to a
range of 0.1 Hz–10 kHz at the formal potential of the redox probe. The significant increase in Ret (DRet = 1.2 6 104). Although
amplitude of the alternate voltage was 5 mV. precipitation of insoluble products on the electrode resulting

This journal is ß The Royal Society of Chemistry 2006 Analyst, 2006, 131, 745–750 | 747
Fig. 3 AFM images show the topological changes on the electrode surface of: (a) the immobilization of anti-HBsAg antibodies; (b) antibody–
antigen complexes after incubation with 100 pg ml21 HBsAg; (c) the sandwich complexes formed after incubation with HRP-labelled anti-HBsAg
antibodies; (d) the precipitation by the HRP-labelled antibody catalysis. The brighter parts correspond to higher features.

748 | Analyst, 2006, 131, 745–750 This journal is ß The Royal Society of Chemistry 2006
from the enzymatic reaction was reported to amplify the EIS data can further be simulated using the Zplot/Zview
impedance signal of biosensors,9,16,17 no report describes the software (Scribner Associates Inc.) and the relevant equivalent
subtle topological changes and height differences of particles circuit in this experiment is shown as the insert of Fig. 4. The
distributed on the gold electrode while employing an circuit includes the ohmic resistance (Rs) of the electrolyte
enzyme label method in the detection of pathogenic solution, Warburg impedance (W), the constant phase element
proteins. Therefore, the results further indicated that the (CPE) and electron-transfer impedance (Ret). The impedance
precipitation by enzymatic catalysis could significantly amplify spectrum of measurement data in this experiment were further
the antibody–antigen interactions and increase the detectable fitted using this equivalent circuit and the fitting spectra are
sensitivity. shown in continuous lines in Fig. 4. As a result, the impedance
values related to Ret could also be determined. Fig. 5 shows a
3.2. Impedance measurement of HBsAg plot of impedance values versus different concentrations of
HBsAg. The target molecules can be detected within the con-
The impedance methods based on HRP-label and AP-label centrations of HBsAg ranging from 10 pg ml21 to 10 ng ml21.
were reported to detect anti-dinitrophenyl antibody7 and Moreover, we can get a good linear relationship between
Escherichia coli O157:H7,9 respectively. In order to explore impedance and concentration of HBsAg ranging from
the possibility of combining the impedance with traditional 10 pg ml21 to 1 ng ml21 with an R2 value of 0.99. The
ELISA methods, HBsAg as a model sample was first used to detection limit of 10 pg ml21 was also obtained through
develop an impedance biosensing method based on enzymatic- comparison of the impedance of the sample with the control
label. Compared with free-label immunological impedance plus double standard deviation, which is more sensitive than
methods,13,18 the introduction of labelled antibodies based the most popular commercially available ELISA testing kit.
on the ELISA scheme could provide higher specificity and The results show that the impedance methods would be used
sensitivity. This merit would be of significance especially for for detection of the gold electrodes array based on the enzyme
real samples analysis such as clinical diagnosis. label techniques.
In this experiment, the anti-HBsAg antibody-modified gold
electrode was incubated with various concentrations of HBsAg 3.3. Detection of multiple antibody–antigen interactions
samples. The resulting impedance spectra are shown as Fig. 4.
The array was further developed to analyse multiple antibody–
It can be seen that the electron-transfer resistance enhanced
antigen interactions. In this experiment, three kinds of
with increasing concentration of HBsAg sample. It implies that
proteins, goat-anti-rIgG, anti-HBsAg antibody and anti-
the more HBsAg molecules bound to the anti-HBsAg anti-
HBeAg antibody, were immobilized on the electrodes array
body-modified electrode, the more precipitate biocatalysed by
through the immobilization frame. In the case of traditional
HRP-labelled antibodies would be deposited on the electrode
electrochemical immunological detection methods, soluble
to further block the interfacial electron-transfer process.
enzymatic products such as hydrogen peroxide were produced
and led to the changes in the amperometric signal. If this
scheme is coupled with multiple biosensing elements, however,
such a soluble enzymatic product would probably diffuse into
neighbouring electrodes and cause detectable cross-talk
signals. In comparison, an insoluble enzymatic product could
probably be coupled with impedance measurement.
In order to study the detection possibility of multiple
antibody–antigen interactions, a series of antigen samples such
as 100 pg ml21 rIgG, 100 pg ml21 HBsAg, 100 pg ml21 HBeAg

Fig. 4 Faradaic impedance spectra that correspond to anti-HBsAg

antibody-modified gold electrodes incubated with different con-
centrations of HBsAg samples, (a) representing the control of BSA
(1 mg ml21) and (b–e) representing HBsAg concentrations of
10 pg ml21, 100 pg ml21, 1 ng ml21 and 10 ng ml21, respectively. A
dotted line and a continuous line represent faradaic impedance
spectrum of a measurement and the fitting spectrum, respectively.
Insert is the equivalent circuit, which represents each component at the
working electrode interface and in the solution during the electro-
chemical reaction in the presence of redox couples. Ret: electron-
transfer impedance; Rs: solution resistor; CPE: constant phase element; Fig. 5 The relationship between the concentration of HBsAg and the
W: Warburg resistance. impedance values.

This journal is ß The Royal Society of Chemistry 2006 Analyst, 2006, 131, 745–750 | 749
 electrochemical signals of antibody–antigen interactions
occurring at the electrode surface. Secondly, the analytical
performance of the present impedance method was further
evaluated. A good linear relationship between the electron-
transfer resistance and HBsAg concentrations ranging from
10 pg ml21 to 1 ng ml21 was found. Finally, multiple antigen–
antibody interaction detection was carried out. The results
show that the present impedance detection based on the
enzyme-label method could not only increase detection
sensitivity, but also prevent a crossover phenomenon on the
array. These advantages show its potential applications in
clinical diagnosis and antibody screening as well as protein–
protein interactions in proteomics research.

Acknowledgements
Fig. 6 Detection of multiple antigen–antibody interactions using
the gold electrodes array; x-axis represents different samples. The This work was supported by Hi-tech Research and
concentration of each constituent was 100 pg ml21. Development Program of China (863 Program, No. 2002-
BA711A11) and National Basic Research Program of China
and the mixture (100 pg ml21 HBsAg + 100 pg ml21 HBeAg) (973 Program, No. 2004CB520804). We thank Sa Zhang
was incubated with the antibody array. The results of (National Center of Biomedical Analysis) for his technical
impedance measurement are shown in Fig. 6. The RIgG support for AFM measurement and analysis of the AFM
sample shows that no cross-talk phenomenon was observed images.
when rIgG was incubated with the electrodes array. In fact, the
insoluble precipitate of AEC produced by HRP-label would References
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750 | Analyst, 2006, 131, 745–750 This journal is ß The Royal Society of Chemistry 2006